Imaging system using multi-mode laser illumination to enhance image quality

ABSTRACT

The quality of image produced by confocal microscopy, and especially scanning laser confocal microscopy, is enhanced especially for images obtained in turbid mediums such as many biological tissues specimens, by reducing speckle from scatterers that exist outside (above and below) the section which is being imaged by utilizing reduced coherence illumination, such as provided by a multi-mode laser. The laser beam is focused to provide its intensity in lobes forming offset spots in opposite (180°) amplitude phase relationship. The lobes are combined in the return light from the section and detected after passing through the confocal aperture of the confocal microscope. Images can be formed from the detected return light. Light from scatterers outside the section of interest, which are illuminated by both of the lobes beams overlap outside the section and interfere, thereby reducing speckle due to such scatterers, and particularly scatters which are adjacent to the section being imaged.

[0001] This application is related to my U.S. patent application Ser.No. 08-966,046, filed Nov. 7, 1997 and provisional application Ser. No.60/072,334, filed. Jan. 23, 1998.

DESCRIPTION

[0002] The present invention related to imaging system which enhanceimage quality be reducing noise which reduces contrast in images,especially images obtained from turbid media, such as encountered inbiological specimens, and especially dermatological tissue whereinkeratin is present. Media, which are turbid, may be characterized byhaving a high RMS refractive index variation and high scattering crosssections.

[0003] The invention is especially suitable for use in confocalmicroscopy and especially in raster scanning confocal microscopes suchas the Vivascope confocal scanning raster microscope sold by LucidTechnologies, Inc. of Henrietta, New York, U.S.A and described in anarticle by M. Rajadhyaksha, et al. entitled “In Vivo Confocal ScanningLaser Microscopy of Human Skin, Melanin Provides Strong Contrast” thatappeared in the Journal of Investigative Dermatology, Volume 104, No. 6,pg. 1 (June 1995) and also the subject matter of an article by M.Rajadhyaksha and James M. Zavislan which appeared on Laser Pocus World,pg. 119 (February, 1996) and in the hand-held scanning laser microscopewhich is the subject matter of U.S. patent application Ser. No.08/650,684 filed May 20, 1996 in the name of James M. Zavislan, et al.The invention is also useful in optical coherence tomography orinterference microscopy.

[0004] It has been discovered in accordance with the invention, that byilluminating a medium with low spatial coherence laser radiation,especially transverse multi-mode radiation and which propagates and inthe TEM₀₁ or higher modes, images obtained from return light from animage plane or section within a specimen, by responding to the intensityof the return light, have reduce image distortion. Distortion producedby scattering sites adjacent to the image plane or section tends to beminimized or at least reduced to a constant value, while optical signalsdue to index variations and other optical activity within the imageplane or section (region of interest) are actually detected. Thus,correlated noise from scatterers, which produces optical distortion andespecially speckle effects in the image, is reduced, thereby enhancingthe quality of the image. The focal region (image plane or section) maybe at the surface of the specimen or embedded in the specimen and theincident light is focused at a laser beam waist into components ofopposite phases. Outside the focal plane (in the section) the componentsoverlap and destructively interfere before detection. Noise due toscattering sites away from the focal region may occur, whether theregion is at the surface or embedded in the specimen. The section beingimaged, especially in imaging of biological tissue, can be of thethickness of a cell, for example, about five microns.

[0005] Regions adjacent to the section of interest may have an abundanceof scatterers, both behind and ahead of the section in the direction ofpropagation of the illuminating beam, which is incident on the section.These potential scattering sources are illuminated by the same opticalfield that illuminate the region of interest. There is a finiteprobability that return light from these scatterers will pass through aconfocal aperture and reach the detector as optical signals from whichthe image of the section of interest is constructed. The spurious returnlight may manifest itself as speckle in the image. The use of multi-modelaser illumination, in accordance with the invention, has been found toreduce such distortion, and especially speckle distortion, therebyproviding additional contrast and enhancing the image quality.

[0006] Confocal microscopes have heretofore used single mode laserswhich propagate usually in the TEM₀₀ mode, in order to obtain a singlecomponent spot or dot in the focal plane. As described in RE 34,214issued Apr. 6, 1993 to Carlsson, the laser beam is focused at a singlespot in the focal plane which is conjugate optically to the confocalaperture. The present invention uses a plurality of spots due to lobes(components) of multimode, preferably TEM₀₁ or higher modes, which lobesare in out of phase amplitude relationship where such modes are focussed(at the laser beam waist-which lies in the focal plane). The lobesoverlap outside the focal plane, thus reducing the spurious, undesirablereturns from scattering sites outside of the focal plane, which definesthe section of the specimen of interest. The above referencedapplications use polarization techniques to shear the beams which, likethe multi-mode illumination, produces spots which are spaced apart inthe focal plane and overlap and cancel spurious reflections (as fromscatters) outside the focal plane, but required polarization prisms andlenses. More specifically my prior applications, Ser. Nos. 08-966046 and60/072,334, referenced above, further enhance image quality in imagingsystems by utilizing circularly polarized beams focused on the imageplane thereby obtaining noise reduction in the image, especially specklenoise which may be attributable to scatterers adjacent to the imageplane. The spots may be laterally offset or vertically offset andprovide different modalities for imaging.

[0007] The noise reduction system described herein also has applicationto optical coherence imaging often referred to as opticalcoherence-domain reflectivity, optical coherence tomograph or opticalcoherence microscopy. (See Schmitt et al, Optical characterization ofdense tissues using low-coherence interferometry, SPIE, Vol. 1889, pps.197-211, July, 1992). In this imaging modality, a low temporal coherencesource is used to illuminate an interferometer with a phase-modulatedreference arm and a sample arm. In the sample arm, a focussing objectivedirects light into a sample, often a turbid biological specimen. Onlylight which is scattered from a depth in the tissue that has equaloptical path as the optical path of the reference arm constructivelyinterferes at the detector to provide an electronic signal thatrepresents the optical signal from the sample. This coherencerequirement eliminates the need for a confocal pinhole to select theimage plan inside the tissue. Optical coherence imaging however, suffersfrom the same deleterious effect of adjacent scatters as does confocalimaging. This effect is reduced, however, by the multi-mode laserillumination and detection system previously described.

[0008] Accordingly, it is the principal object of the present inventionto provide improved imaging systems, and especially imaging systemsusing confocal microscopy, and more especially improved laser scanningconfocal microscopes.

[0009] It is a further object of the present invention to provideimproved confocal microscopes and especially improved laser scanningconfocal microscopes.

[0010] It is a still further object of the invention to provide improvedconfocal laser scanning microscopes which provide images of biologicaltissue, and especially dermatological tissue.

[0011] It is a still further object of the inventor to provide improvedinstruments using optical coherence interferometry.

[0012] Briefly described, a system embodying the invention enablesviewing a section of a medium. Light is received by and returned fromthe section and from sites adjacent to the section. The system utilizestransverse multi-mode laser illumination to provide light which isincident on the medium. This incident illumination is focused in thesection being imaged to provide spots which are spaced from each otherin the plane of the section of interest. The spots are due to the lobesor components of the incident multi-mode laser light which are inopposing (180°) phase amplitude relationship. The lobes overlap outsideof the focal plane, thereby providing inference of light returned fromthe sites (scatterers) adjacent to the section being imaged. The imagemay be constructed in response to the intensity of the return light.

[0013] The foregoing and other objects, features and advantages of theinvention, as well as presently preferred embodiments thereof, willbecome more apparent from reading of the following discussion inconnection with the accompanying drawings in which:

[0014]FIG. 1 is a schematic diagram of a laser scanning confocalmicroscope which embodies the invention;

[0015]FIGS. 1A & B are schematic diagrams of optical arrangements forsynthesizing multimode beams for confocal or optical coherence imaging

[0016]FIG. 2 is a schematic diagram illustrating the processing, in themicroscope of FIG. 1, of the incident multi-mode light and thecollection of the return light from an image section which is shown as afocal plan;

[0017]FIGS. 2A, B and C are plots of the amplitude of the multi-modelaser beam at the beam waist in the focal plan shown in FIG. 2, forTEM₀₁₁ TEM₀₂ TEM₀₃ illumination respectively.

[0018]FIG. 3 is a schematic diagram showing the collection optics in thereturn arm of a confocal microscope system of the type illustrated inFIG. 1 which detects intensity of the return light and enables theconstruction of the image in response thereto; and

[0019]FIG. 4 is a schematic diagram of an optical coherence imagingsystem embodying the invention.

[0020] Referring to FIG. 1, there is shown a confocal laser scanningmicroscope wherein the beam, which is made incident on and illuminates aturbid sample 12, is obtained from multi-mode laser 14, and which in thecase where the microscope is used to image a section of dermatologicaltissue (forming a turbid sample 12), is preferable in the infra-redrange. The incident beam from the laser may be linearly polarized asindicated by the arrow 16. Then, polarizing beam splitter 18 passes theincident beam to scanning optics 20. However, the polarization of theincident beam is not restrictive and any polarization, even circular,may be used.

[0021] The scanning optics provide scanning in an X,Y direction, where Xand Y are coordinates orthogonal to each other in the image plane. Thescanning optics may be an undulating or pivoting mirror and a rotatingpolygon mirror as in the Vivascope laser scanning confocal microscopereferenced above. Orthogonal mirrors may provide the scanning optics, asin the confocal scanning microscope described in the above-referencedpublications. The scanning optics is controlled by a computer controller22 which also collects image data from a photo detector 24 andconstructs the image either on a display, printer or a recorder 26.

[0022] The incident and return beams are deflected by a mirror 28through quarter wave plate toward the sample 12 and pass through anobjective lens system 30 to the focal or image plane in the sample 12.

[0023] The return light from the image plane is again deflected by thescanning optics 20 and deflected by the beam splitter 18 throughdetector optics (a condenser lens system) 34 to the detector. Thedetector optics focuses the light at the center of a confocal aperture36. In order to select the image plan, the objective 30 together withany processing optics 32 (which may be an assembly) is movable undercontrol of the computer control 22 in the Z direction which is adirection perpendicular to the X and Y direction as shown at 40. So fardescribed, except for the processing elements, the confocal laserscanning microscope 10 is similar to that described in the referencedarticle and patent application.

[0024] The multi-mode laser 14 may produce multi-mode TEM₀₁ or higher(e.g., TEM₀₂ or TEM₀₃) modes of propagation by design of its cavity. Thelaser may be a laser diode pumped YAG laser which generates light atabout 1.06 micron wavelength, but instead of as a diffraction limitedsingle-lobed beam, produces a multi-lobed beam. The mirrors at the endsthe cavity or one of them may be cocked away from the confocal axis toenhance the TEM₀₁ mode. Alternatively, a split thin film (λ/2) retardercan be used to generate the TEM₀₁ mode or TEM₀₂ mode outside of thecavity as shown in FIGS. 1A & FIG. 1B. Reference may be had to thefollowing for more information or to the design of a suitable multi-modelaser: O. Svelto, Principles of Lasers, 3RD Edition, Plenum Press, NY &London, 1989 see, especially, pps. 137-206.

[0025] Referring to FIG. 2, the TEM₀₁ mode effectively provides two beamcomponents A and B. The beam components A and B are focused as spots Cand D, respectively in the focal or image plan 58. It will beappreciated that these spots are scanned in X, Y and Z over the imageplan in order to provide optical signals from which the image can beconstructed, after detection by the detector 24, in the computer 22.

[0026] The components are two lobes of TEM₀₁ focused mode. Thecomponents are 1800 out of phase They form an optical beam with twolaterally offset illumination zones which have substantially overlap ofthe two beams in regions away from the beam waist. Thus, scatterersoutside of focus will create a scattered light field with two electricfield modes that are out of phase. These two electric fields will cancelas the collected scattered light is imaged to the confocal aperture 36which is conjugate to the illuminating beam waist. As shown in FIGS. 2Band C, higher modes may be used. In the TEM₀₂ mode are in spaced pairsof about equal and opposite amplitude.

[0027] The light is returned and collected by the objective 30 andcombined. The intensity of light returned from the spots C and D dependsupon the optical reflectance averaged across the spots C and D. Theintensity is the sum of the squares of the intensity of the lightreturned from each spot C and D. Accordingly, the amount of light fromthe image plane which is focused by the condenser 34 and passes throughthe confocal aperture as the optical signal which is detected by thedetector 24, depends upon the effect of the material specimen in thefocal plane.

[0028] Referring to FIG. 3, collection optics of the invention isillustrated. There, a polarizing or leaky beam splitter 60 passes thelaser light beam to the scanning optics. The intensity of the light fromthe scatterers outside of the focal plane in the sum of the intensity ofeach TEM₀₁ component. The return beam is then focused by detector optics34 at the confocal aperture 36 and then detected by the photo detector24. Since the TEM₀₁ components overlap, they interfere and cancel in thecombined beam passing through the confocal aperture 36.

[0029]FIG. 4 shows an optical coherence imaging system with improvedimaging. A low temporal coherence optical source 230 provides transversemulti-mode illumination. The laser 14 or the techniques of FIGS. 1A and1B may be used, but a super luminescent diode or femtosecond laser issuitable. The light therefrom is collimated by lens 235. A linearpolarizer 265 polarizes the incident light. The polarization state isoriented to be in the plane of FIG. 4. The light then passes into beamsplitter 240 which is nominally 50%-50% nonpolarizing beam splitter. Aportion of the light is directed to a reference mirror 250. Referencemirror 250 is actuated by transducer 255, which may be a piezo-electricactuator. This actuation modulates the phase of the reference arm light.

[0030] Light scattered from the two spots inside or on the object iscollected by lens 210 and angularly combined in the objective 210 anddirected towards the beam splitter 240. A portion of the reference andsample light is directed to a photodetector and signal conditioningcircuit 245 which may be a silicon photodiode and amplifier. The portionof the light from both arms incident on the detector that is bothparallel and coherent will interfere in a detection arm terminated atthe detector 245 and produce a phase modulated electric signal whichvaries synchronously with the reference mirror position. The amplitudeof the modulated signal is proportional to the reflectance of thesubject at the point inside the object that has equal optical path asthe reference arm to within the coherence length of the source.

[0031] As with the confocal system described previously, there aresignal contributions from scatterers above and below the surface whichequal path as the reference arm. These scatters will produce specklenoise that interferes with the fidelity of the signal. The scattererswhich are outside the surface of equal optical path will be illuminatedby the overlapping lobes or components. The light from these scatterswill be substantially destructively interfering at the detector becausethe two components have 180° phase difference and illuminate each of thescatterers similarly.

[0032] Controller 260 controls the scan position of the objective lens210 through actuator 225. Controller 260 also controls the position ofactuator 255 which controls the position of the reference mirror 250.The controller collects the signal and decodes it with the positioninformation of the actuators and drives a display or recorder 270.

[0033] From the foregoing description, it should be apparent that therehas been provided an improved imaging system, and especially an imagingsystem which is especially adapted for providing improved confocalmicroscopes and especially laser scanning confocal microscopes and whichis also applicable for optical coherence tomograph or microscopy.Variations and modifications in the herein described system, within thescope of the invention, will undoubtedly suggest themselves to thoseskilled in the art. Accordingly, the foregoing description should betaken as illustrated and not in a limiting sense.

1. A confocal microscope system for viewing of a section of a mediumwhich receives and returns light both from the section and from sitesoutside the section that reduces the quality of an image formed fromsaid return light said system comprising a source of light and confocaloptics including means for projecting light having components ofintensity which are in out of phase relationship and spaced transverselyfrom each other in the section, but are in spatially overlappingrelation outside the section from said outside sites, and said confocaloptics having a confocal aperture at which said return light from saidcomponents is combined for detection, thereby providing for interferenceof light returned from said outside sites and enabling construction ofsaid image in response to said components from said section.
 2. Thesystem of claim 1 wherein said light is provided by a transversemultimode laser source and means are provided for focussing saidcomponents at a plurality of spots of incident light in said section. 3.The system of claim 2 wherein said light is propagated from said sourcein a TEM mode higher than TEM₀₀.
 4. The system of claim 3 wherein saidmode is selected from the group consisting of TEM₀₁ TEM₀₂ TEM₀₃.
 5. Thesystem of claim 1 wherein source is a multi-mode laser which propagatesin the TEM₀₁ or higher modes of propagation.
 6. The system according toclaim 1 further comprising a condenser for providing said return lightand an objective for focusing said spots, thereby providing saidmicroscope for viewing or construction of an image of said section. 7.The system according to claim 2 further comprising a scanner in the pathof said light for scanning said spots with respect to said section. 8.The system according to claim 7 wherein said scanner is an X-Y scanner,where X and Y are orthogonal directions along said section, an objectivefocusing said light at said spots, and said objective being movable in aZ direction orthogonal to said X and Y directions.
 9. The systemaccording to claim 7 wherein said scanner is in the path of saidincident and return light.
 10. The system according to claim lq whereinsaid source is a laser providing a beam of said light which is incidenton said medium sheared in a direction traverse to the direction of saidbeam.
 11. The system according to claim 6 wherein said is a confocalmicroscope has a splitter passing light received by said medium anddeflecting said return light to said condenser, said condenser focusingsaid return light at said confocal aperture.
 12. A scanning confocalmicroscope which comprises a laser providing an incident beam, a beamsplitter, a scanner for scanning an image plan in a specimen section ingenerally orthogonal X-Y directions in said plan, said laser being aplural mode laser providing an intensity distribution having a pluralityof lobes in out of phase relationship, forming spaced spots in a focalplane in said section and overlapping spots outside of said section, andan objective for focusing said spots in said focal plan, a confocalaperture, a photo detector behind said aperture, and optics for focusingreturn light deflected by said beam splitter at said aperture.
 13. Themicroscope according to claim 12 wherein said objective is movabletogether in a Z direction, generally orthogonal to said X-Y directionsthereby selecting different focal plans of said specimen where saidspaced spots are incident.
 14. An optical coherence imaging system whichcomprises a laser source providing light which a low temporal coherenceand transverse multi-mode beam splitter which directs the light fromsaid source into a reference arm and a sample arm incident on an imageplan in a specimen section, a scanner in each sample arm for scanningeach specimen in generally orthogonal directions on said plane, and alsoin said sample arm, an objective having an optical axis for focusingsaid low temporal coherence light at a plurality of spots offset fromeach other in a detection arm to which light is directed by said beamsplitter from said reference and sample arms and means for providingimages in response to interference of light in said detection arm. 15.The system according to claim 14 wherein said objective is movable in adirection generally orthogonal to said orthogonal directions therebyselecting different image planes in said specimen.
 16. The system ofclaim 14 wherein said source is a multi-mode laser propagating saidincident light in TEM modes higher than the TEM₀₀ mode.
 17. The systemof claim 14 wherein said beam splitter is a non-polarizing beamsplitter.